Debranching enzymes and DNA sequences coding them, suitable for changing the degree of branching of amylopectin starch in plants

ABSTRACT

DNA sequences are described which on the codogenic strand code plant debranching enzymes, whose transcripts formed in transgenic plants code new proteins with the enzymatic activity of debranching enzymes which in transgenic plants reduce the degree of branching of amylopectin starch and DNA sequences which on the codogenic strand code plant debranching enzymes, whose transcripts formed in transgenic plants prevent the synthesis of proteins with the enzymatic activity of debranching enzymes, which in the transgenic plants increases the degree of branching of amylopectin starch, as well as plasmids on which these DNA sequences are localized, which can be introduced into plant cells and plants. Also described is a process for the production of plants changed by genetic engineering whose amylopectin starch is modified, and the modified starch obtainable from these plants.

BACKGROUND OF THE INVENTION

The present invention relates to DNA sequences which on the codogenicstrand, code plant debranching enzymes whose transcripts formed intransgenic plants code new proteins with the enzymatic activity ofdebranching enzymes which in transgenic plants reduce the degree ofbranching of amylopectin starch. (The invention also Relates) to DNAsequences which on the codogenic strand code plant debranching enzymeswhose transcripts formed in transgenic plants prevent the synthesis ofproteins with the enzymatic activity of debranching enzymes, which intransgenic plants increases the degree of branching of amylopectinstarch, and also to recombinant plasmids on which these DNA sequencesare localized and which can be introduced into plant cells and plants.

The invention also relates to a process for the production of plantschanged by genetic engineering whose amylopectin starch is modified, andto the modified starch obtainable from these plants.

Polysaccharides such as starch are, alongside along with oils, fats andproteins, essential renewable raw materials from plants.

A decisive factor which stands in the way of the use of renewable rawmaterials is the lack of substances which precisely meet therequirements of the chemical industry in regard to form, structure, orother physico-chemical parameters. In order to make the application ofrenewable raw materials feasible in as many fields of use as possible,it is particularly important to achieve a great material diversity. Inregard to polysaccharides, this means that, for example, as manydifferent forms of starch must be provided as possible. Thisnecessitates considering both strongly branched forms which arecharacterized by a high surface reactivity in their chemical properties,and mildly branched types which are distinguished by a high uniformityof structure. Uniformity of structure is an important prerequisite forhighly efficient reaction control during chemical syntheses.

Although starch is a polymer comprising chemically uniform basiccomponents, the glucose molecules, it is a complex mixture of verydifferent molecule forms which differ in respect to their degree ofpolymerization and the occurrence of branchings of the glucose chains.Starch is therefore not a uniform raw material. In particular, adistinction is drawn between amylose starch, an essentially unbranchedpolymer comprising α-1,4 glycosidically linked glucose molecules, andamylopectin starch, which for its part is a complex mixture ofdifferently branched glucose chains. The branchings come about throughthe occurrence of additional α-1,6 glycosidic linkages.

In typical plants for starch production, such, for example maize orpotato, the two forms of starch occur in a ratio of roughly 25 partsamylose to 75 parts amylopectin.

In regard to the uniformity of a basic substance, such as starch, forits application in the industrial sector, plants are needed which, forexample, contain only the component amylopectin or plants which containonly the component amylose. In regard to the versatility of the rawmaterial starch, plants are needed which show forms of amylopectin withdifferently marked branching. There is thus a great interest in enzymesof the starch metabolism which can modify the degree of branching of thestarch molecules, or in gene sequences which can be used for thegenetically changing plants so as to be able to synthesize differentforms of starch in plants.

It is already known that for certain plant species, for example maize,plant types which contain only amylopectin can be produced bymutagenesis in which individual genes of the plant are inactivated. Forpotato, a genotype which forms no amylose was likewise produced bychemical mutagenesis with a haploid line (Hovenkamp-Hermelink et al.,1987, Theor Appl Genet 75: 217-221). Haploid lines, or the homozygoticdiploid or tetraploid lines developed from them, are not usable inagriculture, however. The mutagenesis technique is not applicable to theagriculturally interesting heterozygotically tetraploid lines, as aninactivation of all copies of a gene is technically not possible becauseof the presence of four different genotype copies. It is known fromVisser et al. (1991, Mol Gen Genet 225: 289) that plant types which formsubstantially pure amylopectin starch can be produced by antisenseinhibition of the gene for the starch granule-bound starch synthetase inpotato.

A branching enzyme of the potato is known from WO 92/14827. This enzymeis known as the Q-enzyme of Solanum tuberosum. It is also known that,with the help of DNA sequences which contain the information for thebranching enzyme of the potato described in WO 92/14827, transgenicplants can be produced in which the amylose/amylopectin ratio of thestarch is changed.

While the occurrence of several Q-enzymes is known for other species,e.g. maize (Singh & Preiss, 1985, Plant Physiol 79: 34-40), it is notknown whether, besides the branching enzyme of the potato known from WO92/14827, other enzymes are involved in the synthesis of branched starchin potato. Besides the Q-enzymes which introduce branchings into starchmolecules, enzymes occur in plants which dissolve branchings. Theseproteins, also known as debranching enzymes, are divided into threegroups according to substrate specifity:

The pullulanases, which besides pullulane also use amylopectin, occur inmicroorganisms, e.g. Klebsiella, and plants. In plants, they are alsocalled R-enzymes. The isoamylases, which do not work with pullulane, butdo with glycogen and amylopectin, likewise occur in microorganisms andplants. An isoamylase of maize is described by Manners & Rowe (1969,Carbohydr. Res. 9: 107), and Ishizaki et al. (1983, Agric Biol Chem 47:771-779) describe an isoamylase of potato. The amylo-1,6-glucosidasesare described in mammals and yeasts and use limiting dextrins assubstrates.

Besides five endo- and two exoamylases, Li et al. (1992, Plant Physiol98: 1277-1284) detected only one debranching enzyme of the pullulanasetype in sugar beet. This enzyme, which has a size of ca. 100 kDa and apH optimum of 5.5, is localized in the chloroplast.

Ludwig et al. (1984, Plant Physiol 74: 856-861) describe a debranchingenzyme from spinach which uses pullulane as a substrate but whichdisplays an activity three times lower upon reaction with amylopectin.

In the case of the agriculturally important starch-storing cultivatedplant the potato, the activity of a debranching enzyme was investigatedin 1951 by Hobson et al. (1951, J Chem Soc 1451). It was demonstratedthat the corresponding enzyme, unlike the Q-enzyme, does not possess achain-extending activity and merely hydrolyses α-1,6 glycosidic bonds.However, it was possible neither to characterize the enzyme moreprecisely nor to describe DNA sequences which code a protein with theenzymatic activity of a debranching enzyme.

To date, no DNA sequences are known which code a protein with theenzymatic activity of a debranching enzyme from plants which, uponintroduction into the plant genome, change the metabolism of the plantin such a way that the degree of branching of the amylopectin starch isincreased or reduced.

SUMMARY OF THE INVENTION

The object of the present invention is to provide DNA sequences

which code debranching enzymes on the codogenic strand, plasmids withwhich these DNA sequences can be introduced into plant cells or plants,plant cells from which whole plants can be regenerated and plants whichmake possible the production of amylopectin starch with an increased orreduced degree of branching.

There are now described the identification and purification ofdebranching enzymes and also peptide sequences of these enzymes andtheir use for the description of DNA sequences which in transgenicplants form transcripts which code proteins with the enzymatic activityof debranching enzymes, or which in transgenic plants form transcriptswhich prevent the synthesis of proteins with the enzymatic activity ofdebranching enzymes and plasmids and plant cells for the production ofthese transgenic plants.

Also described are transgenic plants which contain DNA sequences whichcode proteins with the enzymatic activity of debranching enzymes whichreduce the degree of branching of amylopectin starch and transgenicplants which contain DNA sequences which prevent the synthesis ofproteins with the enzymatic activity of debranching enzymes, whichincreases the degree of branching of amylopectin starch.

a) firstly, proteins with the activity of a debranching enzyme arepurified to homogeneity (see example 1),

b) peptide sequences are established from the purified enzyme by proteinsequencing (see example 2),

c) these peptide sequences are used for the cloning of cDNA sequencesfrom a cDNA library, both immunological and molecular genetic methodsbeing used (see examples 3 and 4) and/or

d) these peptide sequences are used for the cloning of genomic DNAsequences from a genomic library with the help of molecular biologicalmethods (example 5) and finally

e) the DNA sequences from c) and/or d) are introduced into plasmidswhich make possible a transformation of plant cells and the regenerationof transgenic plants (see example 6).

The DNA sequences described by way of example in the examples withreference to the potato (Solanum tuberosum) code for a plant debranchingenzyme which modifies the degree of branching of amylopectin starchnaturally contained in plants in that the degree of branching of theamylopectin starch is increased or reduced as required. Proteinsequences of debranching enzymes with at least one of the followingsequences are coded from the codogenic DNA sequences:

    __________________________________________________________________________    Seq ID No. 1                                                                       Arg Thr Leu Leu Val Asn Leu Asp Ser Asp Asp Val Lys Pro                         1                 5                  10                                   -      Glu Gly Gly Asp Asn Leu Gln                                                15                   20                                                   - Seq ID No. 2                                                                -      Arg Leu Ser Ser Ala Gly Ile Thr His Val His Leu Leu Pro                   1                 5                  10                                   -      Thr Tyr Gln Phe Ala Gly                                                    15                   20                                                   - Seq ID No. 3                                                                -      Gly Ser Glu Val Leu Met His Asp Gly Lys                                    1                5                   10                                   - Seq ID No. 4                                                                -      Ser Pro Ser Glu Ala Asp Pro Val Glu Ile Val Gln Leu Lys                    1                5                   10                                   - Seq ID No. 5                                                                    Asp Cys Ile Gln Val Gly Met Ala Ala Asn Asp Lys                               1                5                   10                                   - Seq ID No. 6                                                                -      Lys Leu Gln Leu His Pro Val Gln Met Asn                                    1                5                   10                                   - Seq ID No. 7                                                                -      Glu Leu Asp Gly Val ValTrp Ser A1a Glu                                     1                5                  10                                    - Seq ID No. 8                                                                -      Ser Leu Leu Asn Ser Leu Ser Thr Glu Lys                                    1                5                   10                                   - Seq ID No. 9                                                                -      Ala Asn Val Glu Arg Met Leu Thr Val Ser Lys                                1                5                   10                                   - Seq ID No. 10                                                               -      Leu Glu Gln Thr Asn Tyr Gly Leu Pro Gln Gln Val Ile Glu                    1                5                   10                                   -      Lys                                                                        15                                                                        - Seq ID No. 11                                                                   Tyr Gly Leu Pro Val Gln Val Phe Glu                                           1                5                                                        - or Seq ID No. 12                                                                Arg Thr Leu Leu Val Asn Leu Asn Ser Asp Asp Val Lys                           1                5                   10                                 __________________________________________________________________________

Transgenic plants with an increased or reduced degree of branching ofamylopectin starch can be produced via a process which is characterizedby the following steps:

a) Production of a DNA sequence with the following part-sequences:

i) a promoter which is active in plants and ensures the formation of aRNA in proposed target tissues or target cells,

ii) a DNA sequence which allows the transcription of RNA which, intransgenic plants, codes a new protein sequence with the enzymaticactivity of a debranching enzyme or which allows the transcription of aRNA which in transgenic plants prevents the synthesis of a protein withthe enzymatic activity of a debranching enzyme,

iii) if necessary, a 3'-non-translated sequence which in plant cellsleads to the ending of transcription and to the addition of poly-Aradicals to the 3'-end of the RNA,

b) transfer and incorporation of the DNA sequence into the genome of aplant cell, preferably using recombinant plasmids and

c) regeneration of intact, whole plants from the transformed plantcells.

Preferably one, more or all of the protein sequences, Seq ID No. 1 toSeq ID No. 12, are contained in the protein sequence of the debranchingenzyme named under ii). Recombinant plasmids according to process stepb) contain the DNA sequences which on the codogenic strands code plantdebranching enzymes or fragments thereof, whereby the transcriptsderived from the DNA sequences in transgenic plants effect the synthesisof new proteins with the enzymatic activity of debranching enzymes whichin the transgenic plants reduce the degree of branching of amylopectinstarch or the transcripts derived from the DNA sequences in transgenicplants prevent the synthesis of endogenous proteins with the enzymaticactivity of debranching enzymes, which in transgenic plants increasesthe degree of branching of amylopectin starch. The latter can beachieved through a cosuppression or by anti-sense RNA (Inouye, 1988,Gene 72: 25-34; Flavell, 1994, Proc. Natl. Acad. Sci. USA 91:3490-3496).

The transgenic plants obtainable through the process with an increasedor reduced degree of branching of amylopectin starch are also thesubject of the invention. Plants to which the process is applied areuseful plants such as e.g. maize, wheat and potato.

The invention also relates to proteins having the enzyme activity of adebranching enzyme, one or more of the sequences, seq ID No. 1 to Seq IDNo. 12 and a molecular weight between 50 kd and 150 kd, especiallybetween 70 kd and 130 kd, above all between 90 kd and 110 kd. Theproteins are from plants, such as Solanum tuberosum.

For the identification of a new DNA sequence containing the informationfor the synthesis of a protein with the enzymatic activity of adebranching enzyme or for the suppression of the formation of anendogenous activity of a debranching enzyme, protein extracts wereobtained from plants, (such as potato plant) by way of example. For thedetection of the enzymatic activity of the debranching enzyme, asdescribed in example 1, a colour test was used. When protein extracts ofpotato plants are separated in non-denaturing, amylopectin-containingpolyacrylamide gels (PAAG), a protein with a starch-modifying activitycan be detected by subsequent iodine dying. While unbranched amyloseforms a blue-coloured complex with iodine, amylopectin produces areddish-violet colouring. In amylopectin-containing PAAGs which turnreddish-violet with iodine, at places at which a debranching activity islocalized, a colour shift to a blue colouring of the gel, occurs as thebranchings of the violet-colouring amylopectin are broken down by theenzyme.

By separating the protein from others with the help of progressiveammonium sulphate precipitation and subsequent affinity chromatographyat immobilized β-cyclodextrin, the protein is purified to homogeneityaccording to the invention. Peptide sequences are determined from thepure protein (see example 2). As a result, peptide sequences of a plantdebranching enzyme are accessible for the first time. The peptidesequences of the debranching enzyme show, in individual areas, a certainhomology to microbial debranching enzymes, but this is not true of alldomains of the protein. The new debranching enzyme from Solanumtuberosum thus represents a previously unknown type of debranchingenzymes.

The peptide sequences of the debranching enzyme serve according to theinvention to identify DNA sequences in plants which code a peptide withthe activity of a debranching enzyme. Immunological processes can beapplied (see example 3) or molecular genetic methods are used (seeexamples 4 and 5).

After the DNA sequences which code a new debranching enzyme areidentified, they can be multiplied in bacteria by cloning into vectorplasmids. Examples of vectors are pBR322, pUC-series, m13mp-series etc.The DNA sequence which codes the new debranching enzyme can be providedwith linkers which permit a simple recloning into other plasmids. Forthe purpose of introduction into plants (see example 6), binary plasmidswhich contain a replication signal, for example, for Escherichia coliand for Agrobacterium tumefaciens can be used preferably, but notexclusively. If these binary plasmids contain T-DNA elements, a transferof the DNA sequence of a new debranching enzyme into the genome ofdicotyledonous plants is particularly simple. Other methods areavailable however, for example transformation with the help of ballisticprocesses which are used for the transformation of monocotyledons (cf.Potrykus, 1991, Ann Rev Plant Mol Biol Plant Physiol 42: 205-225). Toensure an expression of the transferred transgene in genetically changedplants, the cDNA sequence of the new debranching enzyme is fused to apromoter sequence. All the promoters which are active in plants comeinto consideration in principle. Preferably promoters which are activein the starch-storing organs of the plants to be transformed a reused.Thus, in the case of maize, it is the maize granules, whereas in thecase of the potato, it is the tubers.

The tuber-specific B33 promoter (Rocha-Sosa et al., 1989, EMBO J 8:23-29) can be used in particular, but not exclusively, for thetransformation of the potato. For the stabilization of the RNA formed bythe transgene, a termination and polyadenylation signal is also appendedif necessary to the DNA sequence coding the debranching enzyme. This canbe, for example, the termination signal of the octopine synthase genefrom Agrobacterium tumefaciens.

By fusing a promoter, a DNA sequence and, if necessary, a terminationsignal, constructs formed which are integrated into suitable plasmidsfor transformation of plants. These recombinant plasmids are also thesubject of the present invention. The recombinant plasmids are used forthe transformation of plant cells from which whole plants can beregenerated. These plant cells which contain the DNA sequences accordingto the invention are also the subject of the invention. The recombinantplasmids can also be used for the identification of nucleic acidsequences which code debranching enzymes.

As a result of the transfer of a DNA sequence which consists ofpromoter, coding region of a new debranching enzyme andtermination/polyadenylation signal, a transgenic plant is produced inwhich RNA is formed which can serve as a matrix for the synthesis of anew debranching enzyme, or which, through interaction with an endogenousmRNA of a debranching enzyme, suppresses its synthesis. The type of RNAwhich is transcribed by the transgene depends on the orientation of theDNA sequence of the new debranching enzyme relative to the promoter. Ifthe 5' end of the DNA sequence of the new debranching enzyme is fused tothe 3' end of the promoter, a translatable mRNA is formed which servesas a matrix for the synthesis of a protein with the enzymatic activityof a new debranching enzyme. If, on the other hand, the 3' end of theDNA sequence of the new debranching enzyme is fused to the 3' end of thepromoter, an antisense RNA forms which suppresses the translatability ofthe endogenous mRNA of the debranching enzyme.

In the first case, there is an additional enzymatic activity of adebranching enzyme in the plant. The result of this is that the degreeof branching of the amylopectin formed by the transgenic plant isreduced. A starch thereby becomes accessible which, compared with thenaturally occurring type, is distinguished by a more markedly orderedspace structure and an increased uniformity, which has favourableconsequences, for the film-formation properties in particular.

In the second case, an endogenous enzymatic activity of a debranchingenzyme is suppressed. This leads to the formation of markedly branchedstarch in transgenic plants. Markedly branched amylopectin has aparticularly large surface and is thereby suitable as copolymer to aparticular extent. A marked degree of branching also leads to animprovement in the solubility of the amylopectin in water. This propertyis very favourable for certain technical applications. Potato isparticularly suitable for the production of markedly branchedamylopectin while exploiting the DNA sequences, according to theinvention, of the new debranching enzyme, but the application of theinvention is not limited to potato. The modified starch formed in thetransgenic plants can be isolated from the plants or from the plantcells with commonly used methods and processed after purification forthe production of foodstuffs and industrial products. The DNA sequenceswhich code for a debranching enzyme can also be used for the isolationof homologous cDNA or of genomic sequences from other plant species,using standard methods.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the purification of the debranching enzyme from Solanumtuberosum.

In this:

    ______________________________________                                        O =         protein extract of a homogenate of tuber                                        tissue from Solanum tuberosum.                                    Passage = passage through an affinity chromatography                           of the protein extract at immobilized β-                                 cyclodextrin                                                                 β-cyclo dextrin = elution of the affinity chromatography                  with dissolved β-cyclodextrin in the                                     concentrations 1 mg/ml, and 10 mg/ml                                          respectively.                                                                DBE = debranching enzyme from Solanum tuberosum.                              DE = Disproportionating enzyme from Solanum                                    tuberosum.                                                                 ______________________________________                                    

FIG. 2 shows the plasmid pB33-R, which is a derivative of the plasmidpBIN19 (Bevan, M., 1984, Nucl Acids Res 12: 8711-8721).

In this:______________________________________A = DraI/DraI fragment(position -1512 to +14) of the promoter region of the B33 gene ofSolanum tuberosum (Rocha-Sosa et al., EMBO J 8:23-29), B = NotI/NotIfragment of cDNA with the coding region of the debranching enzyme ofSolanum tuberosum in sense orientation to Fragment A. C =Polyadenylation signal of gene 3 of the T-DNA of the plasmid pTiACH5(Gielen et al., EMBO J 3:835-846), nucleotides 11749 to 11939, which wasisolated as PvuII/HindIII fragment from the plasmid pAGV40(Herrera-Estrella et al., 1983, Nature 303: 209-213) and cloned afteraddition of a SphI linker to the PvuII cutting point between the SphIand the HindIII cutting points of the polylinker of pBIN19.______________________________________

FIG. 3 shows the plasmid pB33-R-anti, which is a derivative of theplasmid pBIN19 (Bevan, M., 1984, Nucl Acids Res 12: 8711-8721).

In this:______________________________________A = DraI/DraI fragment(position -1512 to +14) of the promoter region of the B33 gene ofSolanum tuberosum (Rocha-Sosa et al., EMBO J 8:23-29), B = NotI/NotIfragment of cDNA with the coding region of the debranching enzyme ofSolanum tuberosum in anti-sense orientation to Fragment A. C =Polyadenylation signal of gene 3 of the T-DNA of the plasmid pTiACH5(Gielen et al., EMBO J 3:835-846), nucleotides 11749 to 11939, which wasisolated as PvuII/HindIII fragment from the plasmid pAGV40(Herrera-Estrella et al., 1983, Nature 303: 209-213) and cloned afteraddition of a SphI linker to the PvuII cutting point between the SphIand the HindIII cutting points of the polylinker of pBIN19.______________________________________

DETAILED DESCRIPTION OF THE INVENTION Example 1

Identification of a New Debranching Enzyme in Solanum tuberosum

Protein extracts of plants of the species Solanum tuberosum wereobtained from tuber tissue. For this, 820 g tuber tissue are homogenizedin 1500 ml of a buffer comprising 50 mM sodium acetate pH 6.0; 2.5 mM1,4-dithio-DL-threitol; 1.5 mM mercaptoethanol; 0.4 mM PMSF and tracesof sodium bisulphite, sodium sulphite and ascorbic acid (spatula tips ineach case). 50 μl are separated from the homogenate (see trace 1 inFIG. 1) in a PAAG. The gel contains 7.5% acrylamide pH 8.6 which iscrosslinked with methylene bisacrylamide up to a degree of 1:75, plus 1%amylopectin. The buffer system for the electrophoresis containstris/glycine pH 8.9. After the gel run the gel is equilibrated in 50 mMtris/citrate pH 7.0; 2 mM ascorbic acid at 22° C. for 4 hours. Thecolouring of the gel takes place with Lugol's solution for 15 minutes.The result of the colouring is shown in FIG. 1, trace 1. Besides ared-coloured band, which goes back to the activity of an enzymeintroducing branchings (branching enzyme or disproportionating enzyme),a strongly blue-coloured band is to be recognized. The blue colouringcomes about through the enzymatic degradation of α-1,6 glycosidicbranchings of the amylopectin which are responsible for its reddish orviolet colour.

Example 2

Purification of a Debranching Enzyme from Solanum tuberosum andDetermination of Peptide Sequences.

Ammonium sulphate is added, continuously at 4° C. with stirring up to aconcentration of 40% of the saturation concentration, to a proteinextract from tuber tissue of Solanum tuberosum obtained according toexample 1. The partial precipitation of proteins which sets in iscontinued with stirring for two hours, then precipitated proteins areseparated by centrifugation. Ammonium sulphate is added to thesupernatant liquid, as described above, up to a value of 50% of thesaturation concentration, proteins again being precipitated out. Thisprotein fraction is separated by centrifugation and then fractionatedfurther.

After dissolution of the precipitate in 20 ml acetate buffer (seeexample 1) and 12 hours' dialysis against double-distilled water, theprotein solution is subjected to a chromatography. 500 mg protein fromthe fractionated ammonium sulphate precipitation are applied per 30 mlbed volume onto a Sepharose 6B column to which β-cyclodextrin wascoupled. Washing with acetate buffer follows until the eluate displaysno absorption at 280 nm. A protein fraction with low affinity to thestationary phase is then eluted with a β-cyclodextrin solution of 1mg/ml acetate buffer, which is discarded. At a cyclodextrinconcentration of 10 mg/ml acetate buffer, the debranching enzyme ofpotato is eluted (cf. FIG. 1, trace *)

The fraction of the eluate highly enriched with debranching enzyme issubjected to an electrophoresis in a denaturing PAAG in accordance withLaemmli's instructions (1970, Nature 227: 680-685). The now denaturedprotein is cut out from the gel. Peptide sequences are determined bystandard procedures.

The peptide sequences of the debranching enzyme from Solanum tuberosumare reproduced in Seq-ID No. 1 to Seq-ID No. 12.

Example 3

Isolation of cDNA Sequences which Code a Debranching Enzyme of Solanumtuberosum, with the Help of Immunological Methods

The protein purified according to example 2 is used for she immunizationof rabbits. With the help of the serums of immunized rabbits, cDNAlibraries which are representative of transcripts of the tuber tissue ofSolanum tuberosum are screened for cDNA clones which contain sequenceswhich code for the debranching enzyme of Solanum tuberosum. For this,total RNA is prepared from tuber tissue of Solanum tuberosum accordingto Logemann et al. (1987, Anal Biochem 163: 16-20). mRNA, polyadenylatedby standard methods, is prepared from the total RNA, and used accordingto the procedure described by Gubler & Hoffmann (1983, Gene 25: 263) forthe synthesis of cDNA. The cDNA is ligated with commercial EcoRI/NotIadapters and then ligated into the EcoRI cutting point of the expressionsystem lambda ZAPII. After packing of the lambda phage DNA into phageheads, E. coli cells of the strain XL1-Blue are infected and plated outon medium in Petri dishes in a density of 25,000 per ca. 75 cm². Afterca. 3 hours' incubation, nitrocellulose filters steeped in 10 mM IPTGsolution are laid on the lysated bacteria cultures, and removed afteranother 3 hours. The filters are used for an immunological sounding bythe Western blot technique described in example 5. pBluescript plasmidsare obtained by in-vivo excision from the phage species obtained afterthree sounding cycles which contain the cDNA sequence of the debranchingenzyme of Solanum tuberosum. The sequence of the cDNA is determinedusing the method of Sanger et al. (1977, Proc Natl Acad Sci USA 74:5463-5467). The insertion can be isolated by cutting with correspondingrestriction enzymes from the pBluescript plasmid which contains thesequence of the debranching enzyme of Solanum tuberosum and cloned bystandard procedures into binary plasmids with the aim of transformingplants (see example 6).

Example 4

Isolation of cDNA Sequences which Code a Debranching Enzyme of Solanumtuberosum, with the Help of Molecular Genetic Methods

The peptide sequences obtained according to example 2 are used for thederivation of oligonucleotide sequences which, taking account of thedegeneracy of the genetic code, reproduce extracts of the DNA sequenceof the debranching enzyme of Solanum tuberosum. According to the derivedoligonucleotide sequences, synthetic oligonucleotides are synthesized bystandard methods. These are used for the scrutiny of cDNA librarieswhich are representative for transcripts of the tuber tissue of Solanumtuberosum. Firstly, a cDNA library is produced by preparing total RNAfrom tuber tissue of Solanum tuberosum according to Logemann et al.(1987, Anal Biochem 163: 16-20). mRNA, polyadenylated by standardmethods, is prepared from the total RNA, and used according to theprocedure described by Gubler & Hoffmann (1983, Gene 25: 263) for thesynthesis of cDNA. The cDNA is ligated with commercial EcoRI/NotIadapters and then ligated into the EcoRI cutting point of the DNA of thephage lambda ZAP II. After packing of the lambda phage DNA into phageheads, E. coli cells of the strain XL1-Blue are infected and plated outon medium in Petri dishes in a density of 25,000 per ca. 75 cm². Afterca.9 hours' incubation, nylon membranes are laid on the lysated bacteriacultures, and removed after 1 minute. The filters are incubated for 1minute in 250 mM HCl, for 5 minutes in 0.5 M NaOH; 1.5 M NaCl, then for5 minutes in 1 M tris/HCl pH 7.5. After drying and fixing at 80° C. for1 hour, the filters are incubated for 4 hours in a buffer comprising

2 fold SSC (sodium chloride/sodium citrate)

10 fold Denhardt's solution

0.1% SDS (sodium dodecyl sulphate)

5 mM EDTA (ethylene diamine tetraacetic acid)

50 mM di-sodium phosphate

250 μg/ml herring sperm DNA

50 μg/ml tRNA

before the radioactively terminally labelled oligonucleotides are added.After 12 hours' hybridization, the filters are washed in 2 fold SSC/0.5%SDS and then autoradiographed. The temperature for hybridization andwashing of the filters is calculated as follows:

    T+15=16.6×[Na+]+0.41×%GC.sub.OLIGONUCLEOTIDE +81.5-675/length.sub.OLIGONUCLEOTIDE

Suitable oligonucleotide sequences can be calculated from the peptidesequences reproduced in Seq-ID No 1 or Seq-ID No 2 of the debranchingenzyme of Solanum tuberosum. In order to obtain the highest possiblehybridization temperature which guarantees an adequate specifity of thehybrid formation, the longest possible oligonucleotides are to be used.But as the length increases, so does the degree of degeneracy, i.e. thenumber of oligonucleotides with different sequence combinations. Degreesof degeneracy of up to 6000 can be accepted. For the peptide sequencegiven in Seq-ID No 1, the sequence for an oligonucleotide probe oflength 26 bp was derived. The oligonucleotide has a degree of degeneracyof 3072 at a GC content of 61% maximum and 38% minimum. This gives amaximum hybridization temperature of 56° C. The basic sequence for thesynthesis of the probe reads:

    __________________________________________________________________________      Peptide Seq-ID No. 1: Asp Ser Asp Asp Val Lys Pra Glu Gly                                    mRNA: 5' GAU UCN GAU GAU GUN AAA CCN GAA GG                    3'                                                                                                      C AG    C   C       G       G                                     probe: 3' CTA AGN CTA CTA CAN TTT GGN CTT CC                    5'                                                                                                      G TC    G   G       C       C                     __________________________________________________________________________

In three probing cycles, phage species which contain the cDNA sequenceof the debranching enzyme of Solanum tuberosum are isolated and used forthe in-vivo excision of a pBluescript plasmid by standard methods. Asinsertion of a pBluescript plasmid, the sequence of the cDNA isdetermined according to the method of Sanger et al. (1977, Proc NatlAcad Sci 74: 5463-5467). The cDNA is isolated by standard methods(Sambrook et al., 1989, Molecular cloning: a laboratory manual, 2nd Ed.,Cold Spring Harbor Laboratory Press, NY USA) from the pBluescriptderivative after digestion with ECORI or NotI and can be cloned bystandard methods into binary plasmids with the aim of the transformationof plants (see example 6).

Example 5

Isolation of Genomic DNA Sequences which Code a Debranching Enzyme ofSolanum tuberosum, with the Help of Molecular Genetic Methods.

The peptide sequences obtained according to example 2 are used for thederivation of oligonucleotide sequences which, taking account of thedegeneracy of the genetic code, reproduce extracts of the DNA sequenceof the debranching enzyme of Solanum tuberosum. According to the derivedoligonucleotide sequences, synthetic oligonucleotides are synthesized bystandard methods. These are used for the scrutiny of genomic DNAlibraries which represent the genome of Solanum tuberosum.

Firstly, a genomic DNA library is produced according to Liu et al.(1991, Plant Mol Biol 17: 1139-1154). E. coli cells of the strain P 2392are then infected with the phages containing the genomic DNA fragmentsand plated out on medium in Petri dishes in a density of 30,000 per cm².After ca. 8 hours' incubation, nitrocellulose membranes are laid ontothe lysated bacterial lawn, which are removed after a minute. Thefilters are incubated for 2 minutes in 0.2 M NaOH, 1.5 M NaCl; 2 minutesin 0.4 M tris/HCl pH 7.5, then for 2 minutes in 2 fold SSC. Afterdrying, the fixing of the DNA takes place via UV crosslinking. Thefilters are then incubated for 3 hours in a buffer comprising

2 fold SSC (sodium chloride/sodium citrate)

10 fold Denhardt's solution

0.15% SDS (sodium dodecyl sulphate)

5 mM EDTA (ethylene diamine tetraacetic acid)

50 mM di-sodium phosphate

250 mg/ml herring sperm-DNA

before the radioactively terminally labelled oligonucleotides are added.After 12 hours' hybridization, the filters are washed in 0.2 foldSSC/0.1% SDS and then autoradiographed. The temperature forhybridization and washing of the filters is calculated as follows:

    16.6×[Na.sup.+ ]+0.41×[%GC.sub.oligonucleotide ]+81.5-675/length.sub.oligonucleotide -15=T

Suitable oligonucleotide sequences can be calculated from the peptidesequences, reproduced in Seq-ID No 1 or Seq-ID No 6, of the debranchingenzyme of Solanum tuberosum. In order to obtain as high as possible ahybridization temperature which guarantees an adequate specifity of thehybridization, the longest possible oligonucleotides are to be used.However, as the length increases, so does the degree of degeneracy, i.e.the number of oligonucleotides with different sequence combinations.Degrees of degeneracy of up to 10 000 can be accepted. If severalpeptide sequences from a protein are known, oligonucleotides can becalculated accordingly and used jointly in an oligonucleotide mixturefor hybrid formation. This can increase the efficiency of the hybridformation. For the peptide sequence given in Seq ID No. 1, the sequencefor an oligonucleotide probe of length 26 bp was derived. Theoligonucleotide has a degree of degeneracy of 3072 at a GC content of61% maximum and 38% minimum. This gives a maximum hybridizationtemperature of 56° C. For the peptide sequence given in Seq ID No 6, thesequence for an oligonucleotide probe of length 20 bp was derived. Theoligonucleotide has a degree of degeneracy of 384 at a GC content of 55%maximum and 50% minimum. The maximum hybridization temperature to becalculated from this is 60° C. Both oligonucleotide probes are used as amixture at a temperature of 54° C. for hybridization.

The basic sequence for the synthesis of the probes reads:

    __________________________________________________________________________    Peptide Seq ID No. 1: Asp Ser Asp Asp Val Lys Pro Glu Gly                              mRNA: 5' GAU UCN GAU GAU CUN AAA CCN GAA GG 3'                                           C AG    C   C       G       G                                     probe: 3' CTA AGN CTA CTA CAN TTT GGN CTT CC 5'                                           G TC    G   G       C       C                                - Peptide Seq ID No. 6: Ile Gln Val Gly Met Ala Ala                                       mRNA: 5' AUU CAA GU-- GG-- AUG GC--GC   3'                                               C   G                                                                         A                                                                 probe: 3' TAA GTT CAI CCI TAC CGI CG   5'                                                 G   C                                                                         T                                                   __________________________________________________________________________

In three probing cycles, phage species which contain the genomic DNAsequence of the debranching enzyme of Solanum tuberosum are isolated.The genomic DNA insertions are isolated from positive clones by suitablerestriction enzymes and separated by gel electrophoresis. The lambda DNAis separated from the genomic DNA sequence and then isolated by standardmethods (Sambrook et al., 1989, Molecular cloning: a laboratory manual,2nd Edition, Cold Spring Harbor Laboratory Press, NY USA), cloned into apBluescript plasmid and transformed in E coli cells of the strain DH5-α.As the genomic DNA produced according to example 5 has a length 8 kb to15 kb on an average, subfragments with a length of 500 bp to 3.5 kb areproduced using standard methods by means of suitable restriction enzymesand subcloned into pBluescript plasmids. The sequence of the insertedDNA of the different pBluescript plasmids is determined using the methodof Sanger et al. (1977, Proc Natl Acad Sci USA, 74: 5463-5467).

The sequences which code a debranching enzyme of Solanum tuberosum areisolated after restriction digestion with suitable enzymes and can becloned by standard methods into binary plasmids with the aim of thetransformation of plants (see example 6).

Example 6

Construction of Binary Plasmids for the Transformation of Aarobacteriumtumefaciens and for the genetic modification of plants with the help ofAgrobacterium

For the plant transformation, the cDNA which was obtained according toexample 3 or 4 as insertion in pBluescript was recloned into a binaryvector which is derived from pBIN19 (Bevan (1984) Nucl Acids Res 12:8711-8720). Two structures were produced: on the one hand, the plasmidpB33-R, and on the other, the plasmid pB33-R-anti (cf. FIG. 2 and FIG.3). The two constructs contain, as promoter for the expression of atransgene in plants, the B33 promoter of Solanum tuberosum (Rocha-Sosaet al., EMBO J 8: 23-29). Whereas pB33-R contains the cDNA in senseorientation, i.e. leads to the formation of a translatable RNA intransgenic plants, pB33-R-anti represents an antisense structure for theinhibition of the expression of the endogenous gene. The constructs wereproduced as follows:

pB33-R: the promoter of the B33 gene of Solanum tuberosum was cloned asDraI fragment (position -1512 to +14 according to Rocha-Sosa et al.,EMBO J8: 23-39) after degradation of overhanging ends with polymerase IIinto the SacI cutting point of the plasmid pUC19. An EcoRI/SmaIfragment, the promoter region was cloned into the binary vector pBIN19which contains the termination signal of the octopine synthase gene fromAgrobacterium tumefaciens in direct proximity to a polylinker comprisingM13mp19. pB33 was formed in the process. The filled NotI fragment of thecDNA isolated according to example 3 or 4 was cloned into the SmaIcutting point of pB33 in sense orientation relative to the promoter (5'end of the cDNA against the 3' end of the promoter). pB33-R was formedin the process.

pB33-R-anti: the filled NotI fragment of the cDNA according to example 3or 4 was cloned into the binary vector pB33 in antisense orientationrelative to the promoter (3' end of the cDNA against the 3' end of thepromoter). pB33-R-anti was formed in the process.

Corresponding to the constructs pB33-R and pB33-R-anti, plasmids wereconstructed which, instead of the cDNA, contain a genomic DNA sequencewhich codes for a debranching enzyme from Solanum tuberosum. For this,the coding DNA sequences were isolated from the pBluescript plasmidsobtained according to example 5 with the help of suitable restrictionendonucleases, the overhanging ends were filled and the obtained DNAfragments cloned into the SmaI cutting point of pB33 in sense orantisense orientation.

For the transformation of Agrobacterium tumefaciens, the binary plasmidis introduced into the cells by direct transformation according to theHofgen & Willmitzer method (1988, Nucl Acids Res 16: 9877). The plasmidDNA of transformed agrobacteria were isolated according to the method ofBirnboim et al. (1979) Nucl Acids Res 7: 1513-1523 and analysed by meansof gel electrophoresis after suitable restriction cleavage. For thetransformation e.g. of potato plants, 10 small leaves, scarified with ascalpel, of a sterile culture are for example laid in 10 ml MS mediumwith 2% sucrose which contains 30-50 μl of an Agrobacterium tumefaciensovernight culture grown under selection. After 3-5 minutes' lightshaking, the Petri dishes are incubated at 25° C. in the dark. After 2days, the leaves are laid out on MS medium with 1.6% glucose, 2 mg/lzeatin ribose, 0.02 mg/l naphthyl acetic acid, 0.02 mg/l gibberellicacid, 500 mg/l Claforan, 50 mg/l kanamycin and 0.8% bacto-agar. Afterone week's incubation at 25° C. and 3000 lux, the Claforan concentrationin the medium is reduced by half. Further cultivation took place asdescribed by Rocha-Sosa et al. (1989) EMBO J I 8: 29.

The testing of the success of the genetic modification of the plants ispossible through analysis of the total RNA as regards the occurrence ofa mRNA which codes the debranching enzyme (in the case of thetransformation with pB33-R), or as regards the disappearance of theendogenous mRNA in the case of transformation with pB33-R-anti. Theisolation of plant total RNA takes place according to Logemann et al.(1987) Anal Biochem 163: 16-20. For the analysis, 50 μg each total RNAare checked, with the help of Northern blots, for the presence orabsence of the transcript.

To test for the presence of the debranching enzyme in transgenic plants,there is an extraction of total protein from plant tissue and then aWestern blot analysis with the antiserum from rabbits described inexample 3. For this, protein extracts are separated by means of gelelectrophoresis in SDS-PAAG according to molecular weight. After SDSPAAG electrophoresis (PAGE), protein gels are equilibrated for 15-30minutes in transfer buffer for graphite electrodes (48 g/l tris, 39 g/lglycine, 0.0375% SDS, 20% methanol) and then transferred at 4° C. ontonitrocellulose filter with 1.3 mA/cm² for 1-2 hours. The filter issaturated for 30 minutes with 3% gelatine in TBS buffer (20 mM tris/HClpH 7.5; 500 mM NaCl). The filter is then incubated for 2 hours with theantiserum in suitable dilution (1: 1000-10,000 in TBS buffer) at roomtemperature. The filter is then washed for 15 minutes each with TBS,TTBS (TBS buffer with 0.1% polyoxyethylene-(20)-sorbitan monolaurate)and TBS buffer. After the washing, the filter is incubated for 1 hour atroom temperature with alkaline phosphatase-conjugated goat-anti-rabbit(GAR) antibodies (1: 7,500 in TBS). The filter is then washed asdescribed above and equilibrated in AP buffer (100 mM tris/HCl pH 9.5,100 mM NaCl, 5 mM MgCl₂). The alkaline phosphatase reaction is startedby substrate addition of 70 μl 4-nitrotetrazolium (NBT) solution (50mg/ml NBT in 70% dimethylformamide) and 35 μl 5-bromo-4-chloro-3-indolylphosphate (BCIP) (50 mg/ml BCIP in dimethylformamide) in 50 ml APbuffer. After 5 minutes, first signals can be observed as a rule. Forthe determination of the amylose/amylopectin content in the starch oftransgenic potato plants, small leaf pieces with a diameter of 10 mm arefloated for 14 hours under continuous light on 6% sucrose solution. Amarkedly increased starch formation in the small leaf pieces is inducedby this light incubation. After the incubation, the amylose andamylopectin concentration is determined according to Hovenkamp-Hermelinket al. (1988, Potato Research 31: 241-246). The determination of thedegree of branching (α-1,6 glycosidic bonds), of the chain length and ofthe size of the starch granules takes place according to Morrison et al.(1990, Methods in Plant Biochemistry Academic Press Lmtd. 2: 323-352).

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 12                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - Arg Thr Leu Leu Val Asn Leu Asp Ser Asp As - #p Val Lys Pro Glu        Gly                                                                             1               5   - #                10  - #                15              - - Gly Asp Asn Leu Gln                                                                  20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: solanum t - #uberosum                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Arg Leu Ser Ser Ala Gly Ile Thr His Val Hi - #s Leu Leu Pro Thr Tyr      1               5   - #                10  - #                15               - - Gln Phe Ala Gly                                                                      20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: solanum t - #uberosum                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - Gly Ser Glu Val Leu Met His Asp Gly Lys                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: solanum t - #uberosum                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - Ser Pro Ser Glu Ala Asp Pro Val Glu Ile Va - #l Gln Leu Lys              1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: solanum t - #uberosum                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - Asp Cys Ile Gln Val Gly Met Ala Ala Asn As - #p Lys                      1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: solanum t - #uberosum                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - Lys Leu Gln Leu His Pro Val Gln Met Asn                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: solanum t - #uberosum                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - Glu Leu Asp Gly Val Val Trp Ser Ala Glu                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: solanum t - #uberosum                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - Ser Leu Leu Asn Ser Leu Ser Thr Glu Lys                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: solanum t - #uberosum                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - Ala Asn Val Glu Arg Met Leu Thr Val Ser Ly - #s                          1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: solanum t - #uberosum                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - Leu Glu Gln Thr Asn Tyr Gly Leu Pro Gln Gl - #n Val Ile Glu Lys          1               5   - #                10  - #                15               - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: solanum t - #uberosum                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - Tyr Gly Leu Pro Val Gln Val Phe Glu                                      1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: solanum t - #uberosum                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - Arg Thr Leu Leu Val Asn Leu Asn Ser Asp As - #p Val Lys                  1               5   - #                10                                    __________________________________________________________________________

What is claimed:
 1. An isolated DNA sequence as contained in plasmidpB33-R or plasmid pB33-R-anti from potato, wheat or maize, said sequencecoding for a plant debranching enzyme on the codogenic strand, whereinthe transcripts formed by the sequence in a transgenic plant code for aprotein having enzymatic activity of debranching enzymes which reducethe degree of branching of amylopectin starch in the transgenic plant.2. An isolated DNA sequence as contained in plasmid pB33-R or pB33-Ranti from potato, wheat or maize, said sequence coding for a plantdebranching enzyme on the codogenic strand wherein the transcriptsformed by the sequence in a transgenic plant prevent the synthesis ofproteins having enzymatic activity of debranching enzymes, whichincreases the degree of branching of amylopectin starch in thetransgenic plant.
 3. An isolated DNA sequence as contained in plasmidpB833-R or pB33-R anti encoding a protein sequence selected from thegroup consisting of SEQ ID No. 1 to SEQ ID No.
 12. 4. A recombinantplasmid pB33-R or pB33-R-anti comprising at least one DNA sequence frompotato, wheat or maize, said sequence coding a plant debranching enzymeor fragment thereof on a codogenic strand, wherein the transcriptsderived from the DNA sequence in a transgenic plant effect the synthesisof proteins with the enzymatic activity of debranching enzymes which insaid transgenic plant reduce the degree of branching of amylopectinstarch.
 5. A recombinant plasmid pB33-R or pB33-R-anti comprising atleast one DNA sequence from potato, wheat or maize, said sequenceencoding a plant debranching enzyme or fragment thereof on the codogenicstrand, wherein the transcripts derived from the DNA sequence in atransgenic plant prevents the synthesis of endogenous proteins with theenzymatic activity of debranching enzymes, which in said transgenicplant increases the degree of branching of amylopectin starch.
 6. Arecombinant plasmid according to claim 4 wherein said DNA sequenceencodes at least one of the protein sequences selected from the groupconsisting of SEQ ID No. 1 to SEQ ID No.
 12. 7. A plant cell comprisingat least one DNA sequence from potato, wheat or maize according toclaim
 1. 8. An isolated DNA sequence as contained in plasmid pB33-R orplasmid pB33-R-anti from potato, wheat or maize encoding a plantdebranching enzyme wherein a transcript formed by the DNA sequence in atransgenic plant codes for a protein having enzymatic activity of adebranching enzyme which reduces or increases the degree of branching ofamylopectin starch in the transgenic plant.
 9. A method for producing arecombinant protein having the enzymatic activity of a debranchingenzyme, one or more of the peptide sequences SEQ ID No. 1 to SEQ ID No.12, and a molecular weight between 50 kd and 150 kd which comprisesexpressing the isolated DNA sequence as claimed in claim 1 in a cell andsubsequently isolating the recombinant protein from the cell.
 10. Themethod for producing a recombinant protein as claimed in claim 9,wherein the molecular weight is between 70 kd and 130 kd.
 11. The methodfor producing a recombinant protein as claimed in claim 9, wherein themolecular weight is between 90 kd and 110 kd.
 12. The method forproducing a recombinant protein as claimed in claim 9, wherein therecombinant protein is from a potato, wheat or maize plant.
 13. Themethod for producing a recombinant protein as claimed in claim 9,wherein the recombinant protein is from Solanum tuberosum.
 14. Anisolated DNA sequence from potato, wheat or maize encoding a plantdebranching enzyme wherein the DNA sequence hybridizes to the DNAencoding the enzyme in plasmid pB33-R and wherein a transcript formed bythe DNA sequence in a transgenic plant codes for a protein havingenzymatic activity of a debranching enzyme which reduces or increasesthe degree of debranching of amylopectin starch in the transgenic plant.15. An isolated DNA sequence from potato, wheat or maize encoding aplant debranching enzyme wherein the DNA sequence hybridizes to the DNAencoding the enzyme in plasmid pB33-R-anti and wherein a transcriptformed by the DNA sequence in a transgenic plant codes for a proteinhaving enzymatic activity of a debranching enzyme which reduces orincreases the degree of debranching of amylopectin starch in thetransgenic plant.
 16. The isolated DNA sequence of claim 14 wherein atranscript formed by the DNA sequence in a transgenic plant codes for aprotein having the amino acid sequences of SEQ ID No. 1 and SEQ ID No.12 and the enzymatic activity of a debranching enzyme which reduces orincreases the degree of branching of amylopectin starch in thetransgenic plant.
 17. The isolated DNA sequence of claim 15 wherein atranscript formed by the DNA sequence in a transgenic plant codes for aprotein having the amino acid sequences of SEQ ID No. 1 and SEQ ID No.12 and the enzymatic activity of a debranching enzyme which reduces orincreases the degree of branching of amylopectin starch in thetransgenic plant.